Accurate chromosome segregation is essential for the faithful transmission of genetic information. The linkage between sister chromatids allows them to be attached to opposite poles of the mitotic spindle, and the prompt dissolution of this linkage allows chromosome to segregate to the poles at anaphase. This proposal describes experiments in budding yeast cells and frog egg extracts to investigate the nature of the linkage between sister chromatids and the mechanisms that regulate this linkage during the eukaryotic cell cycle. Chromosome separation in living yeast cells will be followed by tagging them with a tandem array of the multiple repeats of Lac operator which is visualized by the binding of a protein fusion between the Lac repressor and green fluorescent protein. This novel technique will be used to determine the timing of sister separation in budding yeast, whether all centromeres in a cell separate synchronously, whether different regions of a chromosome separate at different times, whether chromosome structure influences when sisters separate, and whether specific sequences are required to link sister chromatids to each other. Both yeast and frog extracts will be used to determine the relationship between sister linkage and Cdc2/28-cyclin B-associated kinase (mitosis promoting factor, MPF). Does inactivating MPF induce sister separation when the cyclin proteolysis machinery is inhibited? A genetic analysis of sister linkage and separation will be undertaken by using the Lac operator tagged chromosomes to screen nee collections of conditional mutants for those in which sisters either separate precociously or fail to separate at the onset of anaphase. Standard genetic analysis of these mutants will identify proteins that hold sisters together and regulate this linkage. Finally, changes in protein composition or modification associated with sister separation will be identified by using rapid affinity isolation techniques to isolate and compare chromosomes from metaphase and anaphase. Together these experiments will utilize the genetic tractability of yeast and the complex in vitro reactions that are possible in frog extracts to provide an integrated approach to understanding sister linkage and separation. This information will be directly relevant to understanding the generation of aneuploidy seen in Down syndrome and tumor progression.
